A number of patents and publications are cited herein in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.
Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.
Ranges are often expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment.
This disclosure includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Checkpoint Kinase 2 (CHK2)
The human genome is subject to continuos attack on its integrity, and as a consequence eukaryotic cells possess distinct strategies with which to respond to DNA damage. In this situation, cells respond by activation of cell cycle checkpoints, which either lead to the repair of the damaged DNA or, if the damage is too severe, to activation of cellular processes leading to cell death. A key component of this response is the cell cycle checkpoint kinase Cds1, which was first identified in the fission yeast schizosaccharomyces pombe (see, e.g., Murakami et al., 1995). The human homologue of Cds1 is known as CHK2 and is activated in response to DNA damage by phosphorylation at threonine 68, which requires the PI3-Kinase family member, ATM (see, e.g., Matsuoka et al., 1998; Melchionna et al., 2000). This promotes dimerisation of the protein allowing trans-activation through T-loop exchange (see, e.g., Oliver et al., 2006). Once activated, CHK2 can phosphorylate a number of substrates that regulate cell cycle arrest, DNA repair and cell death. Key substrates include the cell cycle phosphatases, Cdc25A and Cdc25C, which are inactivated through degradation and relocalisation respectively (see, e.g., Pommier et al., 2005). Another important substrate is the p53 protein that is phosphorylated at serine 20, which promotes activation of this important tumour suppressor (see, e.g., Hirao et al., 2000; Chehab et al., 2000). Others include BRCA1, E2F, Plk1 and PML (see, e.g., Pommier et al., 2005).
Inhibition of CHK2 has the potential to offer a number of therapeutic strategies for the treatment of cancer.
All cancers, by definition, have some form of aberrant cell division cycle and frequently the cancer cells possess one or more defective cell cycle checkpoints or harbour defects in a particular DNA repair pathway. These cells are more dependent on the remaining cell cycle checkpoints and repair pathways, compared to normal, non-cancerous cells (where all checkpoints and DNA repair pathways are intact) and their response to DNA damage is frequently a critical determinant of whether they continue to proliferate or activate cell death processes and die. For example, tumour cells that contain a mutant form(s) of the tumour suppressor p53 are defective in the G1 DNA damage checkpoint. Thus CHK2 inhibitors that can abrogate the G2 or S-phase checkpoints (induced by such cancer treatments as ionising radiation or chemotherapeutic anticancer agents) are expected to further cripple the ability of the tumour cell to repair damaged DNA.
Many of the known cancer treatments cause DNA damage by either physically modifying DNA or disrupting vital cellular processes that can affect the fidelity of DNA replication and cell division, such as DNA metabolism, DNA synthesis, DNA transcription and microtubule spindle formation. Such treatments include for example, ionising radiation, which causes DNA strand breaks and a variety of chemotherapeutic agents. A significant limitation to these treatments is drug resistance and one of the most important mechanisms of this resistance is attributed to activation of cell cycle checkpoints, giving the tumour cell time to repair damaged DNA. By abrogating a particular cell cycle checkpoint or inhibiting a particular form of DNA repair by inhibition of CHK2, it may be possible to circumvent tumour cell resistance to these agents and augment tumour cell death induced by DNA damage, thus increasing the therapeutic index of these cancer treatments.
Many tumours already possess activated checkpoint pathway(s), due to intrinsic damaged DNA (see, e.g., Gorgoulis et al., 2005; Bartkova et al., 2005). It may therefore also be possible to administer a CHK2 inhibitor as a single agent and obtain therapeutic activity through inhibition of remaining checkpoint pathway(s) that are in operation.
A major effect of cytotoxic cancer treatments, such as ionising radiation or chemotherapeutic agents, is the death of proliferating cancer cells. However, these agents often have side effects, due to toxicity to normal proliferating tissues. One explanation for this is that cancer cells are often resistant to apoptosis due to defective cell cycle checkpoints. Consequently the doses of a particular cancer treatment that are effective against the tumour may also kill the proliferating normal cells that have intact cell cycle checkpoints and undergo apoptosis. One of the most important signalling pathways involved in activation of cell cycle checkpoints is the p53 pathway. The TP53 gene itself is mutated in 50% of human tumours, whilst other components of this pathway are also found altered in cancer (see, e.g., Gorgoulis et al., 2005; Bartkova et al., 2005). Thus, if p53-dependent apoptosis is temporarily inhibited in normal cells the toxicity/side effects of cancer treatments may be reduced. For example, pifithrin-α a pharmacological inhibitor of p53 function, protects mice from lethal and sub-lethal doses of radiation without causing tumour formation (see, e.g., Komarov et al., 1999). In addition, targeted disruption of CHK2 allows the increased survival of mice exposed to radiation (through resistance to apoptosis) and these animals do not show an increase in spontaneous tumour development compared to the wild-type controls (see, e.g., Takai et al., 2002). Pharmacological inhibition of CHK2 has been shown to have a radioprotective effect on normal human cells (see, e.g., Arienti et al., 2005).
In addition, inhibition of CHK2 alone or in combination with other agents may provide new strategies for treatment or prevention of other diseases, disorders or symptoms thereof in addition to cancer where cell death is associated, such as hypoxia, diabetes, stroke and autoimmune disease.
CHK2 is activated in response to the physiological stress of hypoxia/reoxygenation (see, e.g., Gibson et al., 2005). Loss or inhibition of CHK2 sensitises cells to hypoxia/reoxygenation. Therefore inhibition of CHK2 in this context may have particular therapeutic value in the treatment of solid tumours, which are often hypoxic.